Dihydrofolate reductase from a resistant subline of the L1210 lymphoma. Purification by affinity chromatography and ultraviolet difference spectrophotometric and circular dichroic studies

Gupta, S.V.; Greenfield, N.J.; Poe, M.; Makulu, D.R.; Williams, M.N.; Moroson, B.A.; Bertino, J.R.

Biochemistry 16(14): 3073-3079

1977


ISSN/ISBN: 0006-2960
PMID: 19040
DOI: 10.1021/bi00633a005
Accession: 068518126

Download citation:  
Text
  |  
BibTeX
  |  
RIS

Article/Abstract emailed within 0-6 h
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

Abstract
Dihydrofolate reductase [EC 1.5.1.3] was isolated in 80% yield and purity > 99% from a methotrexate resistant subline of mouse L1210 lymphoma (L1210/MTX) by the use of affinity chromatography. PUrity was established by titrating enzyme activity and protein fluorescence with a stoichiometric inhibitor MTX and confirmed by polyacrylamide gel electrophoresis. The apparent molecular weight calculated from filtration through a Sephadex G-100 column is 21,000 .+-. 3%. This reductase contains 4 tryptophan residues/molecule. The binary complexes of enzyme with folate, dihydrofolate, MTX, NAD+ and NADPH resulted in a changed UV absorption spectra compared to the unmixed components. The changes observed on binding of dihydrofolate, MTX and NADPH in the range 240-400 nm in the spectra were similar to those reported for the reductase of Escherichia coli. This indicates that the chemical environment of the bound substrate, inhibitor and coenzyme may be the same in the 2 enzymes. Absorbance changes at 280 and 290 nm suggest that 1 (or more) tryptophan residues are perturbed on binding of these ligands. The intrinsic circular dichroic (CD) spectra of dihydrofolate reductase from L1210/MTX and E. coli B are different. The backbone ellipticity and aromatic region are quite distinctive. The CD of the enzyme-substrate and inhibitor complexes as well as enzyme-MTX-NADPH ternary complexes exhibit some homologies as compared with the E. coli B enzyme. The ligands may be constrained in similar conformation on the 2 enzymes.